ABSTRACT-PROJECT 3 The type III intermediate filament protein, vimentin is relevant to enhanced directed cell motility. However many of the specific functions vimentin plays in directed cell migration have remained elusive. Our data of the past funding period demonstrate that Vimentin Intermediate Filaments (VIF) can interact with the other cytoskeleton components and the cell-substrate adhesion machinery to stabilize microtubule (MT)-regulated cell polarization and traction orientation, which enhances the persistence in migration directionality. The molecular factors and mechanical mechanisms that mediate these interactions in the context of migrating cells have yet to be revealed. To uncover the molecular mechanism of VIF?s function in stabilizing MT polarization we will further develop our quantitative imaging approaches and analyze possible mechanisms of VIF-MT interaction and VIF-MT cross- linkers (Aim 1). We will also analyze the effects of VIF on actomyosin contractility, adhesion distribution and traction generation using high-resolution traction-force microscopy (Aim 2). The proposed functions of VIF in stabilizing MT polarization and organizing cell traction predict that the turnover of VIF sets the time scale of persistence in cell polarity and directed traction. Hence, in environments where directional cues change faster than the time scale of VIF turnover, the VIF network may generate potentially unfavorable migration inertia. Several kinases, such as PKC and PKA, can phosphorylate vimentin and the phosphorylation increases the solubility of vimentin. We hypothesize that the activation of these kinases at the leading edge in response to changing directional cues disassembles VIF locally to facilitate cytoskeleton reorganization and protrusion forming in the new direction. To test this hypothesis we will correlate local PKC and PKA activation with the rate of VIF disassembly and perturb the VIF response to guidance cues by mutagenizing vimentin?s PKC and PKA phosphorylation sites. We will also quantify the dynamics of reorganization in networks of wildtype vs mutagenized VIF in chemotaxis assays, where the magnitude and time scale of variations in cell external guidance cues can be experimentally controlled. This proposed research plan will enhance our understanding of vimentin?s function in directed migration and produce innovative imaging methods that impact on the cytoskeleton field.